Cells collectively migrate during ammonium chemotaxis in Chlamydomonas reinhardtii

The mechanisms governing chemotaxis in Chlamydomonas reinhardtii are largely unknown compared to those regulating phototaxis despite equal importance on the migratory response in the ciliated microalga. To study chemotaxis, we made a simple modification to a conventional Petri dish assay. Using the assay, a novel mechanism governing Chlamydomonas ammonium chemotaxis was revealed. First, we found that light exposure enhances the chemotactic response of wild-type Chlamydomonas strains, yet phototaxis-incompetent mutant strains, eye3-2 and ptx1, exhibit normal chemotaxis. This suggests that Chlamydomonas transduces the light signal pathway in chemotaxis differently from that in phototaxis. Second, we found that Chlamydomonas collectively migrate during chemotaxis but not phototaxis. Collective migration during chemotaxis is not clearly observed when the assay is conducted in the dark. Third, the Chlamydomonas strain CC-124 carrying agg1−, the AGGREGATE1 gene (AGG1) null mutation, exhibited a more robust collective migratory response than strains carrying the wild-type AGG1 gene. The expression of a recombinant AGG1 protein in the CC-124 strain suppressed this collective migration during chemotaxis. Altogether, these findings suggest a unique mechanism; ammonium chemotaxis in Chlamydomonas is mainly driven by collective cell migration. Furthermore, it is proposed that collective migration is enhanced by light and suppressed by the AGG1 protein.

www.nature.com/scientificreports/ NH 4 Cl (source) were positioned in the dish. Cells were homogeneously distributed across the dish at the start of experimentation when using the Petri Dish assay to assess the chemotactic response of Chlamydomonas; however, the cells were observed to begin migrating towards the source agarose (containing 21 mM NH 4 Cl) within 3 h (Fig. 1, Supplementary Movie S1). By 12 h, the migration towards the source agarose was clearly observed where the cells remained near the source agarose even after 24 h of experimentation.
To validate chemical gradient formation via chemical diffusion, we tracked changes in pH in a dish with a bromophenol blue solution, a pH-sensitive colorimetric dye (Supplementary Movie S1). At 3 h after the setup, the ammonium solution diffused through ~ 50% of the Petri dish and ultimately diffused across most of the dish by the 24 h time point. Additionally, a computational simulation was performed to model the mass transfer (Supplemental Fig. S2). The mass transfer of the tracer dye (bromophenol blue) was found to fit the known model for one-dimensional mass transfer following Fick's Law (Eq. S2), validating the presence of a chemical gradient across the dish for the entire 24 h experiment.
These observations confirmed both the formation of an ammonium gradient in the dish for up to 24 h and the directed cellular migration towards the source. A linear correlation between color intensities and cellular densities was observed for densities of 3 × 10 4 to 3 × 10 6 cells/ml to allow for quantifying directed cellular migration ( Supplementary Fig. S3). Changes in color intensity around the agarose blocks were measured to track changes in the spatially local density of Chlamydomonas cells. The migration of the algal population (defined by the Chemotactic Index or CI) was quantified as a ratio of the color intensity around the agarose containing 21 mM NH 4 Cl to the agarose containing 0 mM NH 4 Cl as defined by: In an experiment, when Chlamydomonas ammonium chemotaxis was assayed under homogeneous light, the cellular density around the source agarose block (containing NH 4 Cl) increased to a CI of 1.49 ± 0.04 at 3 h in three independent experiments. At 6 h, the CI increased to 3.72 ± 3.05 while by the 24 h time point, the CI reached 16.00 ± 3.23 (Fig. 1). Conversely, when the pf14 mutant strain (paralyzed mutant due to lack of radial spokes in cilia, Supplementary Table S1) was assayed under homogeneous light, the CI was less than 1.22 ± 0.03 by 12 h and did not exceed 2.00 after 24 h of observation in three independent experiments (Fig. 1). This suggests (1) CI = Average intensity around source Average intesity around sink  www.nature.com/scientificreports/ that a CI value greater than or equal to 2.00 would represent a positive chemotactic response at any given time using the Petri dish assay regardless of the cell duplication time.

Homogeneous light exposure enhances ammonium chemotaxis in Chlamydomonas.
Previous studies on the light dependency in ammonium chemotaxis in Chlamydomonas have been unclear and inconsistent among published reports 8,22,25 . To elucidate the role of light during ammonium chemotaxis, we first conducted experiments to clarify light dependency. In our assay, we used cultures 3-5 h into their night cycle (nighttime) that were grown in a 250 ml flask with 100 ml of medium containing acetate (TAP medium) for 3 or 4 days in a light:dark (12 h:12 h) cycle (mixotrophic culture). During the assay, the light (about 30 μmol photons·m −2 s −1 ) was continuously exposed to the top of the Petri dish for 24 h (Light), in which the culture medium filled the Petri dish about 2 mm in height. Alternatively, a second assay was conducted without exposing the culture to homogeneous light (Dark). We used the Chlamydomonas strains CC-124, CC-125, and CC-4533, which are often referred to as wild type in the scientific community studying Chlamydomonas, yet have been shown to exhibit genetic variations 7 (Supplementary Table S1). The CC-124 strain carries the AGGREGATE1 gene null mutation (hereafter, agg1 − ) that causes Chlamydomonas to migrate away from a light source (negative phototaxis) 7,26 . The other two strains, CC-125 and CC-4533, carry a wild-type AGGREGATE 1 gene (hereafter, AGG1) and migrate towards or away from the light source depending on light intensity (neutral phototaxis). CC-4533 is a cell wall deficient strain (cw15) used for the insertional mutant library CLiP (Chlamydomonas Library Project) and widely used for studies in Chlamydomonas genetics 27 . Despite the variants exhibiting different genetic properties, all strains exhibited positive chemotaxis responses under homogeneous light exposure (Light, Fig. 2). In contrast, no strains showed a strong chemotactic response in the dark (Dark, Fig. 2). Among the strains tested, CC-124 exhibited the most concentrated populations of cells near the source agarose block under homogeneous light exposure (Light) ( Fig. 2A). To examine whether this strong collection of cells near the source agarose exhibited by CC-124 is due to the directed migration of the cells on the bottom of the Petri dish (i.e., cells adhere to and glide on the surface of the dish 28 ) as the result of negative phototaxis away from the overhead light source in the z direction, we exposed the light from the bottom of the dish. We did not observe any differences in the chemotactic response of CC-124 between the top and bottom light exposures (Supplementary Fig. S4). We also found that pf18, the Chlamydomonas mutant strain that can glide but not swim 29 , is defective in ammonium chemotaxis ( Supplementary Fig. S7), suggesting the most concentrated population of cells near the source agarose in CC-124 is not due to the migration of the cells being driven by gliding on the surface of the dish but due to swimming towards a specific location. We also observed that ammonium chemotaxis occurred similarly in both daytime and nighttime samples when the assay was conducted with continuous light (Supplementary Fig. S5). Furthermore, we discovered that ammonium chemotaxis rarely occurs when Chlamydomonas is cultured autotrophically and assayed in a medium not containing acetate ( Supplementary  Fig. S6). Based on these findings, we performed all remaining experiments, unless otherwise specified, using 10 6 cells/ml of Chlamydomonas cultured with acetate (TAP medium) during the night cycle (nighttime), 3-5 h into the dark on the day:night (12 h:12 h) cycle. www.nature.com/scientificreports/ Ammonium chemotaxis occurs independently from the eyespot, calcium-dependent shift in ciliate, or ammonium uptake. Because we observed that homogeneous light exposure during the assay enhanced ammonium chemotaxis (Fig. 2), we sought to investigate if ammonium chemotaxis could occur in a mutant strain that lacks the eyespot structure (eye3-2, CC-4316) required for phototaxis 30 . We also studied a mutant strain deficient in a calcium-dependent shift in ciliate dominance required for phototaxis (ptx1, CC-2894) 6 . Both mutant strains exhibited a CI value greater than 2.00 after 6 h (Supplemental Fig. S7), indicating positive ammonium chemotaxis. These results suggested that ammonium chemotaxis does not require the same underlying mechanisms algae use during phototaxis. We also examined a mutant strain deficient in ammonium uptake (amt4 − , CC-4042) 31 to examine the possibility that a migratory response to the ammonium gradient was determined by cellular uptake of ammonium as has been shown to be the case in bacteria 14 . Similar to the other mutant strains, CC-4042 exhibited a CI value greater than 2.00 after 6 h (Supplemental Fig. S7), indicating positive ammonium chemotaxis. This result suggests that the cellular uptake of ammonium does not determine the chemotactic response towards the ammonium gradient.
Chlamydomonas exhibits collective cell migration during ammonium chemotaxis. All of the chemotaxis experiments performed using the Petri dish assay across the various Chlamydomonas strains identified that the coefficient of variability with respect to the CI across three independent experiments tended to be constantly high. One possible explanation is that some subpopulations of cells migrate collectively, hindering the continuous and steady accumulation of cells near the source agarose. To investigate the possibility of collective migration in ammonium chemotaxis, we compared migration patterns in a Petri dish during either ammonium chemotaxis or phototaxis. For the phototaxis assay, we placed a Chlamydomonas culture in a Petri dish with no agarose blocks resulting in no chemical gradient. The Petri dish was then covered with a cardboard box in which one side was cut off so that a light gradient was created over the Petri dish ( Supplementary Fig. S8). Chlamydomonas prepared from the same culture were used for both phototaxis and chemotaxis assays. Two Petri dishes, one for the chemotaxis assay and the other for the phototaxis assay, were placed in the same photo box.
To evaluate the migration pattern quantitatively, a pixel cluster in which the highest density of cells was detected in the Petri dish images was tracked with a function of time. Two strains, CC-124 (agg1 − , negative phototaxis strain) and CC-4533 (AGG1, neutral phototaxis strain) were examined (Fig. 3). During phototaxis, an area of the highest density of Chlamydomonas CC-124 was detected near the edge of the Petri dish (the darker side of the light gradient) after 3 h. This high density of cells remained near the edge throughout the duration of the experiment. Similarly, an area with the highest density of Chlamydomonas CC-4533 was detected near the edge of the Petri dish (the brighter side of the light gradient) after 3 h. This high density of cells remained near the edge throughout the duration of the experiment; however, a small portion of the cells did accumulate near the negative side of the gradient at the 12 and 24 h time points. These findings suggest that the cells migrate individually and constantly towards or away from the light source during phototaxis. During chemotaxis, an area with the highest density of CC-124 and CC-4533 was identified near the center of the Petri dish after 3 h. This area then moved towards the source agarose over time, with the highest density area reaching the ammonium source by 24 h. This migration behavior, where individual cells first migrate towards the center of the population and then migrate towards the source as a group, is a hallmark of collective migration often observed in animal cells utilizing specialized molecules to maintain cluster cohesion 18 . Accordingly, we concluded that Chlamydomonas collectively migrates during ammonium chemotaxis. We also concluded that collective migration occurs independently from the genomic status of the AGG1 gene. To observe the collected migration at the microscopic level, we placed the Petri dish in which CC-124 was migrating towards an ammonium source on an inverted transmission microscope. While the movement of the cellular aggregate was observed, the movement of the cilia of aggregated cells was not clearly observed. The movement of the cellular aggregate appeared to occur by the activity of non-aggregated cells propelling the aggregate towards the ammonium source (Supplementary Movie S2).
Chlamydomonas lacking the AGG1 gene enhances ammonium chemotaxis by collective migration. Although collective migration was observed in both CC-124 and CC-4533 (Fig. 3), we observed the collective migration in CC-124 (agg1 − ) more clearly than in CC-4533 (AGG1) under homogeneous light exposure (Fig. 2). To investigate the effect of the AGG1 gene in collective migration, we first conducted ammonium chemotaxis experiments with a low density of cells (10 5 cells/ml) for both CC-124 and CC-4533 because the lower density allowed a clearer observation of the collective migration response compared to the standard density used in the Petri dish assay (10 6 cells/ml) (Fig. 4).
To investigate a potential light-gradient bias against migration towards an ammonium source during the Petri dish assay, the source agarose was placed in three different locations in Petri dishes. Both strains (agg1and AGG1) show collected cells in the center of the Petri dish after 6 h (CC-124) or 12 h (CC-4533) of experimentation independent of the location of the source agarose; however, the collection of cells was more prominent in CC-124. The collected cells were then observed to migrate towards the source agarose over time after the initial clustering event. This result suggests that light-gradient bias does not exist against the collective migration of Chlamydomonas towards the ammonium source. It again further suggests that collective migration occurs independently from the AGG1 gene status in the genome; however, the collective migratory response is more prominent in cells lacking the AGG1 gene. In the experiments conducted in the dark, low-density CC-124 (10 5 / ml) migrated towards the source agarose, although clear collective migration was not visible. On the other hand, we could not identify the low-density CC-4533 migrating towards the ammonium source (Supplemental Fig. S9). These findings suggest that lacking the AGG1 gene enhances ammonium chemotaxis in the dark. To examine the hypothesis that lacking the AGG1 gene enhances collective migration during ammonium chemotaxis, the www.nature.com/scientificreports/  www.nature.com/scientificreports/ Petri dish assay was conducted with transgenic CC-124 in which the recombinant AGG1 protein was expressed through the agg1promoter::AGG1-3xHA (Human influenza hemagglutinin) gene expression cassette 7 . The two independent transgenic strains, named #6 and #13, showed reduced collective migration (Fig. 5, Supplementary Movie S3), supporting the hypothesis that cells lacking the AGG1 gene exhibited enhanced collective cell migration during ammonium chemotaxis.

Discussion
A novel Petri dish assay to study chemotaxis reveals different tactic responses to an ammonium gradient, compared to a light gradient, in Chlamydomonas. A simple method was developed to study Chlamydomonas ammonium chemotaxis in a Petri dish (Fig. 1). This method was also demonstrated to be able to study Chlamydomonas phototaxis (Fig. 3). The materials required for the assay are readily available at local or online stores. It is easy to set up and manipulate light and chemical gradient conditions. Another advantage of this new assay is that it is spatially visible while still able to expose cells to homogenous light in the Petri dishes allowing for the ability to visualize cellular behavior such as collective migration. Prior studies utilized capillary tubes or microfluidic devices to analyze Chlamydomonas chemotaxis 21,23 ; however, in these methods, it is difficult to observe the migration of the entire Chlamydomonas population. We observed collective migration in the Petri dish assay, which was not observed during phototaxis in Chlamydomonas (Figs. 3,  4). One disadvantage of this new approach, which was shared with both the capillary tube method and the microfluidic device, is that a steady-state chemical gradient across the entire dish was not established prior to the initiation of the migration studies. This non-steady concentration gradient could potentially explain the observed high coefficient of variation of the fold changes in the chemotactic index as the cells are responding to the developing gradient (Fig. 2). This suggests that the Petri dish assay may not be suitable for detecting small changes in the chemical gradient at the single-cell level during cellular migration caused by genetic variants or environmental conditions. We observed that Chlamydomonas was very sensitive to a light gradient in the Petri dish assay. Unless homogenous light was exposed during the assay, Chlamydomonas were found to be heavily biased towards the light gradient. This suggests that the phototactic response may be prioritized over the chemotactic response in Chlamydomonas. Another finding is that clear detection of Chlamydomonas migration to an ammonium source agarose block takes a longer time (6-12 h) compared to that towards a light source (< 3 h) in the assay using a 100 mm diameter Petri dish. This slow migratory response partially agrees with the results of the studies using a microfluidic experiment. This study found that it took on average 3 h for Chlamydomonas to cross a 4.6 mm long channel (155 µm wide) during an ammonium chemotaxis 23 . This calculates an average migration speed of ~ 0.3 µm s −1 during chemotaxis in the microfluidic device. Conversely, a previous study estimates that an average migration speed during phototaxis could be as fast as 78 µm s −133 . In our Petri dish assay, assuming Chlamydomonas migrate from one edge to the other edge of the 100 mm Petri dish, the migration speed during chemotaxis was found to be roughly 0.6-1.2 µm s −1 . These observations indicate that the cellular migration www.nature.com/scientificreports/ during ammonium chemotaxis is much slower than during phototaxis. This finding supports the idea that the steering mechanism of ammonium chemotaxis differ from that of phototaxis.

Effects of light, circadian rhythm, and trophic conditions on
Chlamydomonas ammonium chemotaxis in the Petri dish assay. We found that Chlamydomonas migrates towards ammonium in the presence and absence of homogeneous light; however, the migration pattern towards the source ammonium was not as clear without continuous light exposure (Fig. 2, Supplementary Fig. S9). In our conditions, the accumulation and dispersion of the cells around the source and from the sink agarose blocks, respectively, occurred most consistently in experiments under homogeneous light exposure. Chlamydomonas encodes at least eight light receptors (opsin, channelrhodopsin, histidine-kinase rhodopsin, phototropin, cryptochrome, UV resistance locus 8, cytochrome, plastocyanin) besides ones used in the photosynthetic machinery in the chloroplast [34][35][36] . Previous studies suggest that phototropin and cytochrome are involved in the mating of gametes and germination from zygote in Chlamydomonas 37,38 . On the other hand, studies suggest that rhodopsins are involved in phototaxis in Chlamydomonas 39,40 . An additional study suggested that phototropin activation suppresses nitrite chemotaxis during gamete formation in Chlamydomonas 41 . In our assay, Chlamydomonas was exposed to the ammonium gradient while in the vegetative phase. Although further analysis is required, our findings underpin the effect of light during Chlamydomonas ammonium chemotaxis. We wished to identify a Chlamydomonas mutant with a defective ammonium chemotaxis response so that a genetic or molecular component could be revealed in the ammonium chemotaxis signal transduction pathway. However, all of the Chlamydomonas mutants tested in this study maintain a positive ammonium chemotaxis response (Supplemental Fig. S7). Our results suggest a few key points. First, the eyespot, the most upstream component in the signal transduction pathway in the phototaxis mechanism 4 , is not involved in the ammonium chemotaxis signal transduction. Second, the calcium-dependent shift in ciliate dominance required for phototaxis is not involved in the ammonium chemotaxis steering mechanism 6 . Lastly, ammonium uptake achieved by AMT4, the major ammonium transporter in Chlamydomonas 31 , is not involved in defining the cellular response against an ammonium gradient. This is distinct from the mechanisms governing bacterial collective migration in which nutrient uptake regulates the tactic response 14 . Further investigation is required to identify genetic components involved in the signal transduction, including the light and chemical receptors, in ammonium chemotaxis in Chlamydomonas.
We also examined the effect of circadian rhythm and trophic conditions on ammonium chemotaxis. In our culture conditions (12 h:12 h light:dark cycle in a medium containing acetate for 3-4 days before the assay), we did not detect a significant difference in the directed migration between the samples in the daytime (light cycle) or nighttime (dark cycle) (Supplementary Fig. S5). Conversely, when we used the samples cultured in an autotrophic condition (12 h:12 h light:dark cycle in a medium without acetate for 7-8 days before the assay), we did not detect an obvious migratory response towards the ammonium source ( Supplementary Fig. S6). This suggests that the cells may alternate the sensitivity against an ammonium gradient based on the trophic conditions in the culture. A previously conducted capillary tube assay found that Chlamydomonas ammonium chemotaxis was most active during the nighttime and least active during the daytime. In this study, the cells were cultured autotrophically before the assay, and it was conducted without light exposure 7 . We suspect that the disagreement between the previously published study and our findings is due to several differences in experimental conditions, which include the trophic conditions of the culture before the assay, light exposure during the assay, and consideration of collective migration.
Chlamydomonas collectively migrate during ammonium chemotaxis. Chlamydomonas exhibit collective migration during ammonium chemotaxis but not during phototaxis (Figs. 3, 4). In chemotaxis, individual cells first migrate toward the center of the population, suggesting Chlamydomonas may utilize the mechanism used in mammalian cells in which specialized molecules maintain cluster cohesion 17 . Our microscopic observation found an aggregation of cells (Supplementary Movie S2). The movement of the cilia in this aggregate was not clearly observed. This suggests that the physical interaction of the cilium-cilium or cilium-cell may maintain cluster cohesion. Chlamydomonas forms an aggregate, known as a palmelloid, when the cells are exposed to stressors [42][43][44] . However, we believe the aggregate observed during chemotaxis is not a palmelloid because a palmelloid forms a membrane around the aggregate (Supplemental Fig. S10), which we do not observe in the chemotactic aggregate (Supplementary Movie S2). The microscopic observation suggests that the aggregate formed during chemotaxis moves toward a source by non-aggregated individual cells. These nonaggregated cells essentially push the aggregate toward the source (Supplementary Movie S2). Although more analysis is required to understand the underlying mechanism, our findings revealed collective migration and the difference between phototaxis and chemotaxis in Chlamydomonas for the first time.
AGG1 suppresses collective migration in Chlamydomonas while light promotes it during ammonium chemotaxis. CC-124, which carries an agg1 − mutation, showed clearer collective migration than CC-4533 (AGG1) when the Petri dish assay was performed at a low cell density (10 5 cells/ml) (Fig. 4). The low density of the CC-124 cells was shown to respond to the ammonium gradient in the dark, while the lowdensity CC-4533 cells did not (Supplemental Fig. S9). The agg1 − mutant was initially isolated as a phototactic strain that exhibits negative phototaxis behavior 45 . Since then, the product of the AGG1 gene has been considered a component in the phototaxis signaling pathway. Genetic analysis found that the agg1 − mutant in CC-124 abolishes expression of the AGG1 protein due to the insertion of a transposon in the gene 7  www.nature.com/scientificreports/ The AGG1 protein is localized in the mitochondria in Chlamydomonas 7 . Because the molecular function of AGG1 is unknown, it is difficult to address how the different phenotypes of CC-124 observed during phototaxis and chemotaxis are linked at this point. This current work found that CC-124 expressing the recombinant AGG1 protein exhibited suppressed collective migration (Fig. 5, Supplementary Movie S3). This led to the hypothesis that AGG1 may function as a suppressor of collective migration during ammonium chemotaxis (Fig. 6). Light exposure made Chlamydomonas migrate more collectively during ammonium chemotaxis regardless of the AGG1 gene status in the genome (Figs. 2, 4, Supplementary Fig. S9). Based on the findings in this study, we propose that collective migration is the major driving force of ammonium chemotaxis in Chlamydomonas (Fig. 6). Collective migration is promoted by light and suppressed by the activity of the AGG1 protein.
Previous studies indicate that light exposure enhances cilia-mediated adhesion in Chlamydomonas 46 , suggesting that adhesion may be involved in collective migration. Together with our finding that ammonium uptake does not affect tactic behavior, we speculate that the mechanisms governing collective migration in Chlamydomonas are more similar to mammalian cells that utilize the cell adhesion to control the collective migration than to bacteria that utilize nutrient uptake for control. Further analyses, including genetic analysis and single-cell tracking during chemotaxis with a steady-state ammonium gradient, will help to address whether these hypotheses are supported.
Colonization and bet-hedging in ammonium chemotaxis. In this study, we found that Chlamydomonas collectively migrate towards ammonium under homogeneous light exposure, which is suppressed by the AGG1 protein. The advantage of collectively migrating is thought to promote directional migration, helping organisms colonize in their environment faster than if they migrate as single cells 14 . This could explain why Chlamydomonas collectively migrate towards ammonium under homogeneous light exposure because Chlamydomonas can photosynthesize, securing the fixing of the primary molecule, carbon, for their fitness. Chlamydomonas would then seek rapid colonization in an environment where the primary nutrient, nitrogen, is enriched. Without light exposure, bet-hedging strategies, in which the individuals stochastically express maladapted phenotypes (i.e., a cell migrates towards a poorer environment), may be dominated just in case environmental conditions at the source worsen suddenly or light is exposed from other directions 47,48 . AGG1 may function as a safeguard to maintain single-cell migration for the bet-hedging strategy under light exposure conditions.

Methods
Chlamydomonas strains. Chlamydomonas strains used in this study were sourced from the Chlamydomonas Resource Center (http:// www. chlam ycoll ection. org/). Cells were grown in 100 ml of Tris-acetatephosphate (TAP) medium 7 in a 250 ml flask stoppered with a sponge plug under a 12 h:12 h (light:dark, light intensity is ~ 30 μmol photo ns ·m −2 ·s −1 ) cycle at 22 °C on a continuous rotary shaker (110 rpm). The transgenic CC-124 was generated as described previously 7 . When autotrophic conditions were required, the cells were cultured in a minimal medium that did not contain acetate in the TAP medium.
Western blotting and phototaxis assay to confirm the expression of AGG1-3xHA protein. Expression of AGG1-3xHA (Human influenza hemagglutinin) was confirmed by western blotting using an anti-HA antibody and Wakabayashi's phototaxis assay to confirm rescue from negative phototaxis phenotype as described in 7 . www.nature.com/scientificreports/ Petri dish assay. The Petri dish assay for chemotaxis with homogeneous light was performed in a photo box (FotoPro LED 20 × 20 Studio-in-a-Box, Fotodiox Inc.) with a reflective Mylar film (Mylar Diamond Film, TEXALAN) covering all sides. The Petri dish assay for chemotaxis in the dark was performed in incubators without light. The Petri dish assay for phototaxis was performed in the same photo boxes. Cultures in their nighttime (3)(4)(5) h into the dark cycle) were used for all assays 3-4 days after inoculation in the 100 ml culture described above. The cultures were placed in 50 ml centrifuge tubes and centrifuged for 5 min at 3000 rpm. Cells were washed with and resuspended in a TAP medium that did not contain ammonium (TAP-N). The cell number was counted with a hemacytometer after paralyzing the cilia movement by adding 300 mM KHPO 4 at a 1:1 ratio. The cell density was adjusted to 10 6 /ml with the TAP-N medium and used immediately. Ten milliliters of the cell suspension were added to each Petri dish (100 mm in diameter, 15 Fig. S8). All homogeneous light exposure was performed using a white LED light (~ 30 μmol photons·m −2 s −1 ) continuously exposed for the entire duration of the 24 h assay.
Determination of chemotaxis Index. Photos of Petri dishes were taken at the 0, 3, 6, 12, and 24 h time points. The photos were analyzed by Fiji 49 . The photos were converted to 8-bit gray-scale images and inverted. The intensity around the agarose blocks was measured. Typically, an ROI (region of interest) was manually drawn so that the ROI was placed about 1 cm around a source agarose block (containing 21 mM NH 4 Cl) within a Petri dish. The same ROI was copied and used for a sink agarose block (containing 0 mM NH 4 Cl). The ROI was also copied and used in images taken at different time points so that the region where the intensity was measured was consistent during the time course. Background signals were obtained by measuring the intensity outside the Petri dish. These intensity values were imported into Excel (Microsoft Corporation) to calculate the chemotaxis index (CI). The CI was defined as: Computational modeling of mass transfer in a Petri dish. Diffusion of 600 µM bromophenol blue (pH is not adjusted) from a 1.5 wt.% agarose block (20 mm × 20 mm × 2 mm) into the water in a dish was quantified by taking images and analyzed by Fiji. This was accomplished by drawing a straight line from the edge of the source agarose to the bottom of the dish (Fig. S2A, 1 h), and the color intensity profile was plotted. The measured intensity was directly correlated to the concentration of dye diffused through the water. Using the known dimensions of the agarose block inside the dish, the measurement scale was defined for every image to convert between pixels in the image and the distance (in cm) in the dish. Images from 1, 4, 8, and 24 h time points were used for the analysis to approximate the diffusion coefficient of bromophenol blue. The mass transfer of chemicals is described by Fick's law as follows as previously characterized by Sung et al. 24 : where C is the concentration of the dye (µM), u is the velocity of the fluid responsible for the convective mass transfer and is equal to zero in the case of this experiment (cm/s), and D is the diffusion coefficient of the molecules in the solution (cm 2 /s). The concentration gradient present in the dish can be predicted using a solution of Eq. S1 using the error function below: (2) CI = Average intensity around source Average intesity around sink www.nature.com/scientificreports/ where C 0 is the initial (source) concentration (µM), x (cm) is the distance, and t (s) represents time. A custom Python code was used to fit the experimental line scan data (Fig. S2A) to Eq. S2; however, since the collected data is measurements of the dye absorbance intensity, concentrations (C and C 0 ) in Eq. S2 were replaced with intensity (I and I 0 ). This is a common approach used to model mass transfer of dyes in water 50 . I 0 was assumed equal to the highest measured intensity in the dish which corresponds to position immediately outside of the agarose block at the 1 h time point. Diffusion coefficients were found for the four timepoints and the average value was used to model the diffusion of bromophenol blue. This diffusion coefficient was approximated to be 1.42 × 10 −4 (cm 2 /s), which showed an accurate alignment between the experimental and simulated data and validated that Equation S2 was a good fit for the mass transfer in the Petri dish (Fig. S2B). The approximated diffusion coefficient was used to model the diffusivity of bromophenol blue through the dish at all experimental time points (Fig. S2C).
The trends and gradients predicted by the model accurately match what was observed in the experimental data (Fig. S2A), confirming the diffusion of bromophenol blue released from the agarose block through the water in the Petri dish. The diffusion coefficient predicted by the experimental data does deviate from the published diffusion coefficient of bromophenol blue in water (4.4 × 10 −6 ) 51 . This difference can be attributed to the image processing and analysis approach. Since the experimental measurements are of absorbance intensity, and the collected data is being fit to a model originally designated for concentration values, it can be a factor in the overapproximation. Moreover, it is known that although the dye concentration and the corresponding absorbance have a linear correlation as described by the Beer-Lambert law, intensity and absorbance are described through an exponential correlation. As such, high concentrations of the dye being used to visualize the mass transfer resulted in greater light intensities when compared to the actual concentration of the dye itself, resulting in the observed variation in the diffusion coefficient.

Microscopy observation of chemotaxis and phototaxis.
Videos of the Petri dishes were captured at Video observation of chemotaxis in the transgenic strains. Images were captured every 5 min for 24 h to create a time-lapse video.

Data availability
All data generated or analyzed during this study are included in this published article and the Supplementary Information.